SOCS3 blocks the inhibitory effect of CD33 on proliferation. (A) CD33WT stable Ba/F3-SOCS3 cells seeded at a density of 1 × 105 cells/mL were cultured in the presence or absence of tetracycline (4 μg/mL). Trypan blue exclusion assay determined the viability of the cells at 24-hour intervals. **P < .005 Student t test (B) CD33WT stable Ba/F3-SOCS3 cells were cultured in the presence or absence of tetracycline for 48 hours. They were lysed, immunoprecipitated, and immunoblotted with α-Flag. (C) CD33WT stable Ba/F3-SOCS3 cells seeded at a density of 1 × 105 cells/mL were cultured in the presence or absence of tetracycline. They were incubated with α-CD33 (IC7/1) and cross-linked with GAM. Fresh antibody was added at 24 hours. Trypan blue exclusion assay determined the viability of the cells at 24-hour intervals. **P < .005. (D) CD33WT and CD33Y340F/Y358F stable Ba/F3-SOCS3 cells seeded at a density of 1 × 105 cells/mL were cultured in the presence or absence of tetracycline. They were incubated with α-CD33 (IC7/1) and cross-linked with GAM. Fresh antibody was added at 24 hours. MTT (10 μL; 0.5 mg/mL) was added to 100 μL cell culture and incubated at 37°C for 2 hours at 24-hour intervals. The cells were centrifuged at 235g for 3 minutes and the supernatant was removed. DMSO (200 μL) was added and incubated for 10 minutes at 37°C. Plates were read at OD570 using a microplate reader. *P < .05. Error bars represent SD from triplicate samples.
